Despite a great deal of research, a complete understanding of the actions of general anesthetics is still not available. The objective of this research is to advance our understanding of isoflurane, with the hope of gaining insights into all anesthetics. Isoflurane, a halogenated volatile anesthetic, is thought to produce anesthesia by depressing central nervous system function. Many anesthetics, including isoflurane, are thought to modulate and/ or directly activate GABAA receptors. Isoflurane is also known to have effects on other channels and receptors. In a set of preliminary studies we observed that isoflurane, at clinically relevant concentrations, dramatically inhibited the neurotransmitter release machinery in PC12 cells. Etomidate and propofol also inhibited the release machinery in PC12 cells, suggesting that inhibition of neurotransmitter release may be an important general action of anesthetics. Because neurotransmitter release mechanisms are strongly conserved between PC12 and neurons we hypothesize that anesthetics interfere with the neuronal neurotransmitter release machinery and this may represent an important site of action for anesthesia in intact animals. The experiments outlined in this application will be carried out in cultured hippocampal neurons, PC12 cells and mutant mice. This proposal has three goals. First, we will determine whether isoflurane interferes with neurotransmitter release in hippocampal neurons at clinically relevant concentrations. Next, the anesthetic site of action will be identified. We start by examining syntaxin 1A and unc-13, but other sites including SNAP- 25, synaptobrevin and synaptotagmin 1, will be investigated as well. Details about why these sites were selected are provided in this application. Once neurotransmitter release machinery targets are identified they will be mutated in order to suppress the interaction between isoflurane and the target. This information will be used to design knock-in mice with the same mutations, which will permit us to determine, using a battery of physiological and behavioral tests, whether these animals still respond to isoflurane (or other anesthetics) or whether their responses to the anesthetics are altered.Project Narrative: Isoflurane is a widely used volatile anesthetic. Our goal is to find out how isoflurane produces anesthesia. In particular, we will explore the effect on the neurotransmitter release machinery, as our preliminary data suggests this may be a critical site of action for isoflurane and other anesthetics. This may allow for the design of new more beneficial drugs in the future.